用于实体瘤早期治疗应答评估的超灵敏 cfDNA 片段组学检测

Introduction: Early identification of treatment response remains a major clinical challenge, as imaging lacks the spatial and temporal resolution required to measure the therapy response. Liquid biopsy (Lbx)-based fragmentomics offer a minimally invasive, tumor-agnostic approach to capture dynamic changes in tumor burden and can serve as an early surrogate marker for response. Here, we present the analytical validation of OncoAlibrex, a highly sensitive, NGS independent cfDNA fragmentomics assay for therapy response monitoring (TRM), and demonstrate its clinical utility in patient samples. Methods: Analytical validation was conducted to evaluate various assay parameter using cfDNA extracted from healthy plasma and cancer patient samples (n =140). cfDNA fragments were quantified using multiplexed qPCR on ABI 7500 thermal cycler. Primers and probes were designed based on high copy number Alu and SVA retrotransposon represented across the genome. cfDNA fragment distributions at >80 bp, >105 bp, and >265 bp to detect tumor relevant shifts linking to tumor burden was detected using fragment-specific primer pairs. Therapy response was predicted using an integrated algorithm developed to interpret cfDNA fragments distribution. Results: Standard cfDNA samples, and cfDNA isolated from healthy volunteers (n = 10) and cancer patients, were analyzed in triplicate across multiple days, operators, and reagent lots. Intra-assay variability was ≤1 % for ct value and ≤13 % for the ratiometric quantity, while inter-assay precision showed <2.0% ct variability. Excellent linearity (R² ≥ 0.99) and qPCR efficiencies (>98%) were demonstrated across 0.6 pg to 20 ng/µL of ccfDNA. Analytical sensitivity parameters per CLSI were: limit of blank = 0. 37 pg/µL (>80 bp, ctDNA specific), 0. 01 pg/µL (>105 bp, ctDNA specific), 0.021 pg/µL (>265 bp, non-tumor specific); limit of detection = 0. 85 pg/µL (>80 bp), 0.17 pg/µL (>105 bp), and 0.074 pg/µL (>265 bp). Fragmentation pattern of patient-derived ccfDNA was enriched with shorter fragments (80-105 bp) compared to healthy individuals. The cfDNA specific fragmentation pattern of cancer patients showed high progression score with 100 % specificity to indicate therapy non-response. Conclusions: OncoAlibrex assay detected tumor-specific cfDNA fragmentation pattern indicative of therapy resistance with very high sensitivity, specificity, and accuracy. This assay can be used for TRM during the early phase of the treatment and MRD detection post-treatment independent of the sequencing-based Lbx assays.